miRNA-200c-3p deficiency promotes epithelial-mesenchymal transition in triple-negative breast cancer by activating CRKL expression

Epithelial-mesenchymal transition (EMT) plays an important role in malignant progression of Triple-negative breast cancer (TNBC). Many studies have confirmed that miRNA-200c-3p is related to EMT. And we found that it is involved in the regulation of EMT, but the exact mechanism is unclear. CRKL is highly expressed in a variety of tumors and plays a role in EMT. In this study, the potential targets of miRNA-200c-3p were searched in miRPathDB, Targetscan and PicTar. And there are 68 potential targets at the intersection of the three databases. Then, bioinformatics and text mining performed by Coremine Medica, and found that among 68 potential targets, CRKL has the strongest correlation with EMT in TNBC. Therefore, we speculated that miRNA-200c-3p involvement in EMT might be related to CRKL. To verify miRNA-200c-3p inhibits the malignant phenotype of TNBC by regulating CRKL, RT‒PCR, western blotting, Clonal formation assays,CCK-8 proliferation assays, transwell invasion assays, Luciferase reporter assay and nude mouse transplantation tumor assay were performed. In this study, we found that miRNA-200c-3p is under-expressed and EMT-related genes are up-regulated in TNBC, and miRNA-200c-3p can inhibit cancer cell proliferation, invasion and the expression of EMT-related genes and proteins in TNBC. Further research confirmed that miRNA-200c-3p could inhibit EMT by inhibiting the expression of CRKL that directly combining CRKL gene.


Introduction
Triple-negative breast cancer (TNBC) refers to breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR) and proto-oncogene HER-2 by immunohistochemical examination of cancer tissue.This type of accounts for 10.0-15% of all pathological types of breast cancer [1,2].It has special biological behavior and clinicopathological characteristics, and the prognosis is worse than other types.Typically exhibits aggressive behavior, including earlier recurrence, metastasis, high mortality and frequently chemotherapy resistance.Although conventional treatments like surgery and radiotherapy have achieved considerable progress in clinic, the 5-year survival rate of TNBC patients remains

Cell proliferation assay
The cells viability assay was executed with the Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan).In short, cells were seeded in 96-well plates and incubator for 24 h.The CCK-8 reagent (10 μL) was added to each well, then cells were incubated at 37 °C for 1 h.Absorbance was measured at 450 nm using an Multiskan Spectrum reader (Thermo Scientific, USA).

Cell migration and invasion assay
Transwell (Corning, MA) was used to analyze cell migration as previously described [23].Briefly, the undersurface of the upper chamber was coated with 10 μg/mL of collagen I. Cells were suspended in a density of 10 6 cells/mL and 100 μL of cell suspension was added into the upper chamber of each well.Cells were allowed to migrate for 24 h, cells on the undersurface of the upper chamber were then stained and counted under a phase contrast microscope.

Wound healing assay
When the cells confluence reached about 80-90%, the 200 μL sterile pipette tip was used for scratching the layers.Then, the scratched cells were washed away by phosphate buffer saline (PBS; Vicmed, China).2% FBS was added into the fresh medium.The culture dishes were placed into the incubator in the same environment as the cell culture.After 24 h incubation, the scratches were captured with a microscope.MShot Image Analysis System was applied for images.

Colony formation
Cells were constructed to the single cell suspension for counting, with 500 cells in 60 mm culture medium.After 12-16 days culture process, the cells were washed 3 times with PBS, then fixed with paraformaldehyde for 15 min, and finally stained with crystal violet for 15 min.Number of clones with > 50 cells was determined under microscope (Olympus Corp., Tokyo, Japan).

Real-time polymerase chain reaction analysis
Total RNA was extracted from cells with Trizol reagent (Takara Bio, Japan), following the manufacture's protocols.The quantity of extracted RNA was determined using the Nanodrop 2000 spectrophotometer (NanoDrop Products, Wilmington, Del.).RNA was reverse transcribed to cDNA using a Prime Script RT reagent Kit (Takara).The mRNA expression was normalized to that of β-actin.Polymerase chain reaction amplification was performed using SYBR Premix Ex Taq (Takara).Amplification and qRT-PCR measurements were carried out using the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, Calif.).The average of genes was normalized to β-actin as an endogenous housekeeping gene.

Western blot assay
Equivalent amounts of protein (30 μg per lane) were loaded and separated by 8%-12% SDS-PAGE gels, and transferred PVDF membrane.After the transfer was completed, the membrane was blocked 30 min and then incubated overnight at 4° with specific primary antibodies as follows: antibodies against E-Cadherin, Cytokeratin, Vinmentin, N-Cadherin and CRKL were obtained from Abcam, β-actin was purchased from Biyuntian Biotechnology Co., LTD.After that the membranes were washed and incubated with secondary antibody for 1 h at room temperature.The bands of proteins were visualized using the ECL Plus system (GE Healthcare, Little Chalfont, United Kingdom).Image J (Scion Corporation) was used to quantify the optical density of each band.

Nude mouse transplantation tumor assay
Nude mice (Shanghai Slack Laboratory Animal CO.,LTD) were injected subcutaneously with 5*10 6 cells/200 μL (MDA-MB-231).Tumor sizes were measured every 4 days after subcutaneous injection.After 1 weeks, total of 1 OD Chol-agomiR-NC or Chol-agomiR-200c-3p was injected at 3 different spots on each established tumor.The mice were sacrificed by cervical dislocation under anesthesia, and tumors were removed for the volumes measurement after 3-4 weeks.

Statistical analysis
Data were presented as Mean ± SD and analyzed by GraphPad Prism 6 software (GraphPad Software, USA).The t-test and one-way ANOVA were employed to analyze continuous variable.The value of P < 0.05 was considered to have statistical significance.All experiments were performed at least thrice.

miRNA-200c-3p is under-expressed and EMT-related genes are up-regulated in TNBC
RT-PCR was performed to measure the expressions of miRNA-200c-3p in TNBC and matched tumor-adjacent tissues.Results indicated that the levels of miRNA-200c-3p in TNBC tissues were lower than those in matched adjacent tissues (P < 0.01, Fig. 1A).In TNBC, Vimentin, N-Cadherin mRNA levels were highly expressed and E-Cadherin, Cytokeratin mRNA levels were down-regulated (Fig. 1B-E), As the same time, the results of western blotting were consistent with those of RT-PCR (Fig. 1F), which indicated that EMT is enhanced in TNBC.

miRNA-200c-3p can inhibit cancer cell proliferation and invasion
miRNA-200c-3p mRNA level in normal breast cell lines and TNBC lines were performed by RT-PCR, and we found that miRNA-200c-3p decreased in TNBC lines.It is more obvious in MDA-MB-231 and BT20 (Fig. 2A), so we used BT20 and MDA-MB-231 for our follow-up experiments.To determine how miRNA-200c-3p effected TNBC cell growth, invasiveness and migration capability, we performed CCK-8, clone formation assay, cell wound scratch assay and transwell assay on TNBC cells.In a growth period of 48 h, miRNA-200c-3p-mimic inhibited cell growth obviously compared to control group (Fig. 2B, C).Cell wound scratch assay showed that miRNA-200c-3p mimic could be significantly inhibited the migration of tumor cells (Fig. 2D, E).Treatment of miRNA-200c-3p mimic resulted in invasion inhibition in TNBC lines compared to control group (Fig. 2F, G).Clone formation assay showed that miRNA-200c-3p mimic could be significantly inhibited clone formation assay tumor cells (Fig. 2H, I).These results demonstrate that miRNA-200c-3p specifically inhibits TNBC cell proliferation and invasion.

miRNA-200c-3p can inhibit the expression of EMT-related genes and proteins in TNBC
To investigate whether miRNA-200c-3p effect the expression of EMT-related genes and proteins of EMT in TNBC, western blotting and RT-PCR were performed to measure the expressions of EMT-related genes and proteins.We found that miRNA-200c-3p-mimic increased the expression of E-cadherin, cytokeratin (Fig. 3A, B) and decreased the expression of vimentin and N-cadherin (Fig. 3C, D).The results of western blotting were consistent with those of RT-PCR (Fig. 3E).These results indicated that miRNA-200c-3p can inhibit the expression of EMT-related genes and proteins in TNBC, and Loss of miRNA-200c-3p can promote EMT of TNBC.

Up-regulation of CRKL promotes the malignant phenotype of TNBC
CRKL has been proved to be an oncogene, which plays a role in promoting tumor invasion and metastasis in various tumors involved in immune escape and EMT.In this study, the potential targets of miRNA-200c-3p were searched in miRPathDB, Targetscan and PicTar.And there are 68 potential targets at the intersection of the three databases (Fig. 4A).Then, bioinformatics and text mining performed by Coremine Medica, and found that among 68 potential targets, CRKL has the strongest correlation with EMT in TNBC (Fig. 4B).Further detection of CRKL expression in TNBC showed that CRKL was highly expressed in cancer tissue (Fig. 5A, B).To verify the effect of CRKL, we knock down CRKL and then western blot detected the knock down of CRKL (Fig. 5C).CCK-8 assay confirmed that CRKL knockdown could inhibit the activity of TNBC Cells (Fig. 5D).In the same time, Transwell assay and clonal formation showed that knockdown of CRKL can inhibit cells proliferation and invasion (Fig. 5E, F).Western blot assay showed that E-Cadherin and Cytokeratin protein expression were increased after CRKL was knocked down, and vimentin, N-Cadherin were decreased, indicated that CRKL promotes EMT of TNBC (Fig. 5G).TNBC accounts for 10-15% of all breast cancer styles [1,2].TNBC is easy to invade and metastasize, leading to poor prognosis and high mortality in patients.While the great improvement has been made towards treating and preventing TNBC with different methods such as chemotherapy, radiation therapy, and surgical operations but metastasis is still remaining the challenge due to the limits of effective treatment and early detection.The breast cancer likely to form metastasis in bone, lung, and liver.Therefore, it is very necessary to search for its invasion and metastasis mechanisms in TNBCs, and exploring intervention measures to improve the prognosis of TNBC is imperative.The EMT is an important foundational process for tissue repair and organ development, and its abnormal activation has been proven to be a key step in tumor progression, distant metastasis, and drug resistance [24,25].In tumors, EMT is associated with the tumour stemness, invasion, metastasis and resistance to treatment [26][27][28][29][30], and which is a key step in TNBC cell metastasis.The occurrence of invasion and metastasis involves a variety of genes and signal transduction pathways, among which the roles of miRNAs is receiving increasing attention at home and abroad [31].
In recent years, miRNA as an effective biomarker or potential therapeutic target has received more and more attention.More and more studies have displayed that miRNA closely related to EMT affect the prognosis of tumors that through complex mechanisms.At present, a large number of research have shown that the miRNA plays a key role in pathogenesis, development, invasion and metastasis of breast cancer [32][33][34].It has also been confirmed that the miR-200 family members can inhibit EMT by directly target the transcriptional regulators ZEBl and ZEB2 [10][11][12]35].However, whether EMT can be regulated by miRNA-200c-3p through other means has not been fully elucidated.
In this study, we found that miRNA-200c-3p lower expression and EMT activated abnormally in TNBC than matched tumor-adjacent tissues.We then tested it in the cell line and found that the results were consistent with those in the tissue.These results suggest that miRNA-200c-3p may play a role as a tumor suppressor in the development of TNBC, which will provide an important theoretical basis for small-molecule drug targeted therapy of breast cancer and the formation of a new pathway for clinical practice.
In order to determine the role of miRNA-200c-3p in TNBC and the effect of it on EMT, we conducted follow-up experiments to verify its function in TNBC.CCK-8 and transwell assay showed that miRNA-200c-3p-mimic significantly inhibited cell proliferation and cell invasion ability.To determine whether miRNA-200c-3p can inhibit EMT in TNBC, miRNA-200c-3p-mimic was exposed to TNBC cell lines, RT-PCT and western blot assay were performed to detect he expression of EMT-related genes and proteins in TNBC, displaying that miRNA-200c-3p-mimic Significantly upregulated the genes and proteins of E-cadherin and cytokeratin, and down-regulated the genes and proteins of vimentin and N-cadherin.Subcutaneous tumor transplantation in nude mice also confirmed that c can inhibit tumor growth and invasion.RT-PCT and western blot assay were performed to detect he expression of EMT-related genes and proteins in tumor of nude mice, displaying that miRNA-200c-3p.Significantly upregulated the genes and proteins of E-cadherin and cytokeratin, and down-regulated the genes and proteins of vimentin and N-cadherin.Which indicated that miRNA-200c-3p could inhibit EMT.
However, the site of action of miRNA-200c-3p still needs to be determined.To predict the target genes of miRNA-200c-3p, miRPathDB, Targetscan and PicTar database were used to identify targets.The target genes of the three databases were intersected, and there were 68 potential targets in total.Furthermore, bioinformatics and text mining performed by Coremine Medical confirmed that CRKL was significantly associated with EMT and malignant phenotype.To further confirm the effect of CRKL on TNBC, the CRKL gene was knocked down.And we found that knockdown of CRKL gene can inhibit the proliferation, invasion and EMT of TNBC.In order to verify the regulatory effect of miRNA-200c-3p on CRKL, the miRNA-200c-3p was overexpressed and interfered.Then, western blot was performed to detected CRKL protein expression level, and found that miRNA-200c-3p-mimic decreased CRKL protein expression level, as the same time miRNA-inhibitor promoted the expression of CRKL protein level.Luciferase reporter assay shows that miRNA-200c-3p can bind directly to CRKL mRNA, and then decreased CRKL protein expression level.Hence, we determined that miR-200c-3p can Inhibit the expression of CRKL through directly combining CRKL gene, and inhibit EMT, proliferation and invasion of TNBC.

Conclusion
These results indicate that the loss of miRNA-200c-3p activates EMT by activating CRKL, and then promoting invasion and metastasis of TNBC.
Author contributions Ff N and Wn M performed assays.Ff N and Qf Z participated in data analysis and manuscript revision.J Y, Qf Z participated in manuscript revision.Ff N, J Y, designed, conceived of the study, and drafted the manuscript.All authors read and approved the final manuscript.

Fig. 1
Fig. 1 The expression of miRNA-200c-3p, EMT-related genes and proteins in cancer and matched tumor-adjacent tissues.A The expression of miRNA-200c-3p in cancer tissues.B The expression of E-cadherin mRNA in TNBC tissues detected by RT-PCR.C The expression of Cytokeratin mRNA in TNBC tissues detected by RT-PCR.D The expression of Vimentin mRNA in TNBC tissues detected by RT-PCR.E The expression of N-cadherin mRNA in TNBC tissues detected by RT-PCR.F The expression of E-cadherin, Cytokeratin, Vimentin, N-cadherin proteins in TNBC tissues detected by western blotting.The values are presented as the means (SD), **p < 0.01 versus control group.All experiments were repeated three times

Fig. 3 Fig. 4
Fig. 3 Effects of miRNA-200c-3p on TNBC cells EMT.A The expression of E-Cadherin mRNA level.B The expression of Cytokeratin mRNA level.C The expression of Vimentin mRNA level.D The expression of N-Cadherin mRNA level.E. The protein expression of E-Cadherin, Cytokeratin, Vimentin, N-Cadherin and β-actin.The values are presented as the means (SD), **p < 0.01 versus control group.All experiments were repeated three times

Fig. 5 Fig. 6
Fig. 5 The expression of CRKL in tumor tissue and the effect of CRKL on the proliferation, invasion and EMT of TNBC.A Expression of CRKL mRNA in TNBC tissue.B Expression of CRKL protein in TNBC tissue.C The protein expression after CRKL knockdown was detected by western blot.D The changes of cell viability after CRKL knockdown were detected by CCK-8.E Transwell assay to verify the effect of si-CRKL on TNBC cells invasion.F Effect of CRKL knockdown on cell clonal formation.G The EMT-associated proteins expression after CRKL knockdown were detected by western blot

Fig. 7
Fig. 7 The effect of miRNA-200c-3p on CRKL and the effect of miRNA-200c-3p on EMT in tumors of nude mice.A Expression of miRNA-200c-3p mRNA.B Expression of CRKL mRNA.C Western blot detected the effect of miRNA-200c-3p on CRKL protein level.D Expression of N-cadherin mRNA.E Expression of Vimentin mRNA.F Expression of E-cadherin mRNA.G Expression of Cytokeratin mRNA.H The EMTassociated proteins were detected by western blot.The values are presented as the means (SD), **p < 0.01, *p < 0.05 versus control group.All experiments were repeated three times